Quantifying Brain-death, Transplant Physiology and Immunosuppression on Porcine Muscle-Derived Stem Cell Expansion
Anil Chaturvedi, MS1, Samuel Boas, BS1, Mohamed Awad, MD1, Corinne Wee, MD1, James Reynolds, PhD2, Anand Kumar, MD1.
1University Hospitals Cleveland, Cleveland, OH, USA, 2Case Western Reserve University, Cleveland, OH, USA.
Background: Muscle-derived stem cells (MDSCs) are robust mediators of tissue regeneration that have been shown, in the literature, to exhibit multipotency in murine models. We hypothesize that immunosuppression, transplant physiology, and brain-death (BD) negatively affect the stem cell-niche in vascularized composite allografts (VCA) and may affect tolerance/rejection. Our study aim was to evaluate the growth of MDSCs under these conditions in a porcine VCA model.
Methods: Muscle samples were harvested from the porcine hind limbs before and after inducing brain-death. Samples were then harvested from the VCA of another immunosuppressed pig and from the contralateral hindlimb of the recipient 7 days after transplantation. MDSC populations were isolated using a modified collagen-sorting preplate technique. Imaging was obtained at various time points (t=0-10 days). Images were analyzed for confluence using ImageJ, statistical analysis was performed using Mathematica.
Results: Time to confluence (t = 3, 5, 7, 10 days) between pre-BD and post-BD MDSC populations was significantly different (20%, 38%, 75%, 81% vs. 7%, 17%, 31%, 39%; p<0.01). Time to confluence (t = 2, 4, 6, 8, 10 days) between immunosuppressed recipient and allograft MDSC populations was significantly different (3%, 6%, 13%, 45%, 55% vs. 7% 11%, 39%, 53%, 71%, respectively; p=0.02). At t=10 days, mean confluence between pre-BD and immunosuppressed recipient MDSC populations was significantly different (81% vs. 55%; p < 0.01); similarly, mean confluence between pre-BD and allograft MDSC populations was also significantly different (81% vs. 71%; p = 0.01). At t=10 days, mean confluence between post-BD and immunosuppressed recipient MDSC populations was significantly different (39% vs. 55%; p<0.01).
Conclusion: Brain-death and immunosuppression significantly impair MDSC expansion and mitosis based on time to confluence; however, transplantation may ameliorate the negative effects of immunosuppression by acting as a surrogate for trauma and inflammation. Brain-death physiology appears to have the greatest impact on MDSC expansion in our study. Further studies will focus on immune modulation and scaffold growth in porcine MDSC populations. 
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